Cruickshank W H, Kaplan H
Biochem J. 1972 Dec;130(4):1125-31. doi: 10.1042/bj1301125.
A competitive labelling method (Kaplan et al., 1971), using tritiated 1-fluoro-2,4-dinitrobenzene as the labelling reagent, is described for determining the ionization constants and reactivities of individual histidine residues in proteins. When this method was applied to the two histidines of alpha-chymotrypsin, histidine-57 was found to have pK(a) 6.8 and a reactivity ten times that of alpha-N-acetyl-l-histidine. Histidine-40 had pK(a) 6.7 and a reactivity approximately six times that of alpha-N-acetyl-l-histidine. Between pH7.5 and 8 the reactivities of both histidines decrease simultaneously to approximately that of alpha-N-acetyl-l-histidine. The high reactivities of the histidines are attributed to hydrogen bonding, which increases the nucleophilicity of the imidazole ring. The sharp decrease in reactivity between pH7.5 and 8 is attributed to a conformational change that disrupts the hydrogen bonding by these residues. The reactivity data support the proposal of a charge-relay mechanism involving histidine-57 (Blow et al., 1969), which makes serine-195 more nucleophilic but indicates that this system is fully operative only in the enzyme-substate complex.
本文描述了一种竞争标记法（卡普兰等人，1971年），该方法使用氚化的1-氟-2,4-二硝基苯作为标记试剂，用于测定蛋白质中各个组氨酸残基的电离常数和反应活性。当将此方法应用于α-胰凝乳蛋白酶的两个组氨酸时，发现组氨酸-57的pK(a)为6.8，反应活性是α-N-乙酰-L-组氨酸的10倍。组氨酸-40的pK(a)为6.7，反应活性约为α-N-乙酰-L-组氨酸的6倍。在pH7.5至8之间，两个组氨酸的反应活性同时下降至约α-N-乙酰-L-组氨酸的反应活性。组氨酸的高反应活性归因于氢键，氢键增加了咪唑环的亲核性。pH7.5至8之间反应活性的急剧下降归因于构象变化，该变化破坏了这些残基之间的氢键。反应活性数据支持了涉及组氨酸-57的电荷中继机制的提议（布洛等人，1969年），该机制使丝氨酸-195更具亲核性，但表明该系统仅在酶-底物复合物中完全起作用。
